This thesis aims to increase the understanding of human osteoarthritis pathophysiology by developing reliable biomimetic ex vivo human osteochondral explant models and focussing on the role of... Show moreThis thesis aims to increase the understanding of human osteoarthritis pathophysiology by developing reliable biomimetic ex vivo human osteochondral explant models and focussing on the role of osteoarthritis-relevant triggers (mechanical stress) and interacting genetic factors for developing treatment targets. Human aged joint tissues were collected in the Research in Articular Osteoarthritis Cartilage (RAAK) biobank. To add knowledge of the osteoarthritis pathophysiological processes, aged human ex vivo osteochondral explants were subject to three osteoarthritis-relevant triggers, being inflammation, hypertrophy and injurious mechanical stress. Next, knowledge on early initiating processes occurring in mechano-pathology was investigated by applying RNA-sequencing to cartilage of aged human osteochondral explants subjected to mechanical stress. In addition, to show that the human osteochondral explant model could also be used for genetic interaction studies, we investigated expression of the osteoarthritis risk gene MGP in relation to rs1800801 genotypes. By combining information from RNA-sequencing datasets of cartilage and bone with osteoarthritis-relevant triggers in cartilage and bone explants we investigated the role of MGP and vitamin K in osteoarthritis. Lastly, the injurious mechanical explant model was exploited to determine the effectivity of inhibiting the osteoarthritis risk gene DIO2 by iopanoic acid treatment either by burst or prolonged release from PLGA-PEG nanoparticles. Show less
Houtman, E.; Tuerlings, M.; Riechelman, J.; Suchiman, E.H.E.D.; Wal, R.J.P. van der; Nelissen, R.G.H.H.; ... ; Meulenbelt, I. 2021
Background Failing of intrinsic chondrocyte repair after mechanical stress is known as one of the most important initiators of osteoarthritis. Nonetheless, insight into these early mechano... Show moreBackground Failing of intrinsic chondrocyte repair after mechanical stress is known as one of the most important initiators of osteoarthritis. Nonetheless, insight into these early mechano-pathophysiological processes in age-related human articular cartilage is still lacking. Such insights are needed to advance clinical development. To highlight important molecular processes of osteoarthritis mechano-pathology, the transcriptome-wide changes following injurious mechanical stress on human aged osteochondral explants were characterized. Methods Following mechanical stress at a strain of 65% (65%MS) on human osteochondral explants (n(65%MS) = 14 versus n(control) = 14), RNA sequencing was performed. Differential expression analysis between control and 65%MS was performed to determine mechanical stress-specific changes. Enrichment for pathways and protein-protein interactions was analyzed with Enrichr and STRING. Results We identified 156 genes significantly differentially expressed between control and 65%MS human osteochondral explants. Of note, IGFBP5 (FC = 6.01; FDR = 7.81 x 10(-3)) and MMP13 (FC = 5.19; FDR = 4.84 x 10(-2)) were the highest upregulated genes, while IGFBP6 (FC = 0.19; FDR = 3.07 x 10(-4)) was the most downregulated gene. Protein-protein interactions were significantly higher than expected by chance (P = 1.44 x 10(-15) with connections between 116 out of 156 genes). Pathway analysis showed, among others, enrichment for cellular senescence, insulin-like growth factor (IGF) I and II binding, and focal adhesion. Conclusions Our results faithfully represent transcriptomic wide consequences of mechanical stress in human aged articular cartilage with MMP13, IGF binding proteins, and cellular senescence as the most notable results. Acquired knowledge on the as such identified initial, osteoarthritis-related, detrimental responses of chondrocytes may eventually contribute to the development of effective disease-modifying osteoarthritis treatments. Show less
Houtman, E.; Hoolwerff, M. van; Lakenberg, N.; Suchiman, E.H.D.; Zwaag, E.V.V. van der van der; Nelissen, R.G.H.H.; ... ; Meulenbelt, I. 2021
Introduction: Likely due to ignored heterogeneity in disease pathophysiology, osteoarthritis (OA) has become the most common disabling joint disease, without effective disease-modifying treatment... Show moreIntroduction: Likely due to ignored heterogeneity in disease pathophysiology, osteoarthritis (OA) has become the most common disabling joint disease, without effective disease-modifying treatment causing a large social and economic burden. In this study we set out to explore responses of aged human osteochondral explants upon different OA-related perturbing triggers (inflammation, hypertrophy and mechanical stress) for future tailored biomimetic human models.Methods: Human osteochondral explants were treated with IL-1 beta (10 ng/ml) or triiodothyronine (T3; 10 nM) or received 65% strains of mechanical stress (65% MS). Changes in chondrocyte signalling were determined by expression levels of nine genes involved in catabolism, anabolism and hypertrophy. Breakdown of cartilage was measured by sulphated glycosaminoglycans (sGAGs) release, scoring histological changes (Mankin score) and mechanical properties of cartilage.Results: All three perturbations (IL-1 beta, T3 and 65% MS) resulted in upregulation of the catabolic genes MMP13 and EPAS1. IL-1 beta abolished COL2A1 and ACAN gene expression and increased cartilage degeneration, reflected by increased Mankin scores and sGAGs released. Treatment with T3 resulted in a high and significant upregulation of the hypertrophic markers COL1A1, COL10A1 and ALPL. However, 65% MS increased sGAG release and detrimentally altered mechanical properties of cartilage.Conclusion: We present consistent and specific output on three different triggers of OA. Perturbation with the pro-inflammatory IL-1 beta mainly induced catabolic chondrocyte signalling and cartilage breakdown, while T3 initiated expression of hypertrophic and mineralization markers. Mechanical stress at a strain of 65% induced catabolic chondrocyte signalling and changed cartilage matrix integrity. The major strength of our ex vivo models was that they considered aged, preserved, human cartilage of a heterogeneous OA patient population. As a result, the explants may reflect a reliable biomimetic model prone to OA onset allowing for development of different treatment modalities. Show less
Aims In mitral valve prolapse (MVP), leaflet thickening has recently been suggested to be due, in addition to a myxomatous degeneration, to the presence of a superimposed tissue (SIT), defined as... Show moreAims In mitral valve prolapse (MVP), leaflet thickening has recently been suggested to be due, in addition to a myxomatous degeneration, to the presence of a superimposed tissue (SIT), defined as an additional fibrous layer on top of the original leaflet. The mechanisms of SIT formation are currently unknown. We hypothesized that SIT formation would result from excessive leaflet stress and we used a unique ex vivo model to assess the correlation between leaflet remodelling and the type and location of mechanical stress and to elucidate the mechanisms underlying SIT formation.Methods and results Human diseased mitral valves (MVs; n = 21) were histologically analysed for SIT formation and original leaflet thickening. The SIT comprised of various compositions of extracellular matrix and could reach more than 50% of total leaflet thickness. Original leaflet and SIT thickness did not show significant correlation (r= -0.27, P=0.23), suggesting different regulatory mechanisms. To study the role of the mechanical environment on MV remodelling, mouse MV were cultured in their natural position in the heart and subjected to various haemodynamic conditions representing specific phases of the cardiac cycle and the MVP configuration. SIT formation was induced in the ex vivo model, mostly present on the atrial side, and clearly dependent on the duration, type, and extent of mechanical stress. Specific stainings and lineage tracing experiments showed that SIT comprises of macrophages and myofibroblasts and is associated with the activation of the transforming growth factor-beta and bone morphogenetic protein signalling pathways. Migration of valvular interstitial cells and macrophages through breakages of the endothelial cell lining contributed to SIT formation.Conclusions Mechanical stresses induce specific cellular and molecular changes in the MV that result in SIT formation. These observations provide the first insights in the mechanism of SIT formation and represent an initial step to identify potential novel and early treatment for MVP.[GRAPHICS]. Show less
The heart is built for life-long uninterrupted function, supporting blood flow through the organism. During life, the organism as well as its organs will become challenged by exogenous and... Show moreThe heart is built for life-long uninterrupted function, supporting blood flow through the organism. During life, the organism as well as its organs will become challenged by exogenous and endogenous stresses that should be coped with. To be able to adapt to stresses such as altered loading conditions, changes in myocardial perfusion and exposure to toxic agents, the heart possesses ample capabilities. This thesis deals with the question how cardiac cells react to different stresses in an attempt to adapt to the new circumstances. The effect of mechanical stress as well as integrin stimulation on cardiac cells was examined and described. Besides we investigated the reaction of cardiac cells to DNA damage caused by either ionizing radiation or UV-irradiation. The studies reported in this thesis confirm that cardiac cells, both myocytes and fibroblasts have the capacity to respond adequately to exogenous stresses. The responses that have been studied in detail demonstrate that these cells possess systems that sense the stress, transmit its message to the cell interior, and alter gene expression, leading to appropriate reactions. Show less
